Process for producing influenza vaccine

ABSTRACT

A process for producing a split influenza virus preparation or subunit influenza preparation comprising the steps of: (i) providing a whole virus preparation; (ii) splitting the whole virus preparation in the presence of a first detergent; (iii) adding t-octylphenoxypolyethoxyethanol (TRITON X-100™) to the resulting split virus preparation; and (iv) filtering the split virus preparation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 13/503,859 filed 25 Apr. 2012, which is the US National Stage ofInternational Application No. PCT/EP2010/066083, filed 25 Oct. 2010,which claims benefit of priority to U.S. Provisional Application No.61/329,230, filed 29 Apr. 2010, which applications are incorporatedherein by reference in their entirety. This application also claimsbenefit of the filing date of GB 0918830.1, filed 27 Oct. 2009.

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BACKGROUND

Field of the Invention

The present invention relates to process for making influenza antigenssuitable for use in vaccines and pharmaceutical compositions comprisingsaid antigens.

Description of the Related Background

Influenza viruses are one of the most ubiquitous viruses present in theworld, affecting both humans and livestock. Influenza results in aneconomic burden, morbidity and even mortality, which are significant.

The influenza virus is an RNA enveloped virus with a particle size ofabout 125 nm in diameter. It consists basically of an internalnucleocapsid or core of ribonucleic acid (RNA) associated withnucleoprotein, surrounded by a viral envelope with a lipid bilayerstructure and external glycoproteins. The inner layer of the viralenvelope is composed predominantly of matrix proteins and the outerlayer mostly of host-derived lipid material. Influenza virus comprisestwo surface antigens, glycoproteins neuraminidase (NA) andhaemagglutinin (HA), which appear as spikes, 10 to 12 nm long, at thesurface of the particles. It is these surface proteins, particularly thehaemagglutinin that determine the antigenic specificity of the influenzasubtypes. Virus strains are classified according to host species oforigin, geographic site and year of isolation, serial number, and, forinfluenza A, by serological properties of subtypes of HA and NA. 16 HAsubtypes (HI-HI 6) and nine NA subtypes (N1-N9) have been identified forinfluenza A viruses [Webster R G et al. Evolution and ecology ofinfluenza A viruses. Microbiol. Rev. 1992; 56:152-179; Fouchier R A etal. Characterization of a Novel Influenza A Virus Haemagglutinin Subtype(H16) Obtained from Black-Headed Gulls. J. Virol. 2005; 79:2814-2822).Viruses of all HA and NA subtypes have been recovered from aquaticbirds, but only three HA subtypes (H1, H2, and H3) and two NA subtypes(N1 and N2) have established stable lineages in the human populationsince 1918. Only one subtype of HA and one of NA are recognised forinfluenza B viruses.

At unpredictable intervals, novel influenza viruses emerge with thehaemagglutinin antigen, of a totally different subtype from strainscirculating the season before. Here, the resulting antigens can varyfrom 20% to 50% from the corresponding protein of strains that werepreviously circulating in humans. This phenomenon, called “antigenicshift” can result in virus escaping ‘herd immunity’ and establishingpandemics. In other words, an influenza pandemic occurs when a newinfluenza virus appears against which the human population has noimmunity.

During a pandemic, antiviral drugs may not be sufficient or effective tocover the needs and the number of individuals at risk of influenza willbe greater than in interpandemic periods, therefore the development of asuitable vaccine with the potential to be produced in large amounts andwith efficient distribution and administration potential is essential.Improved processes are also important to maximize production of antigenfor seasonal (interpandemic) influenza vaccines, as the populationgenerally ages and the need for influenza vaccines increases.

The present invention addresses this need.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides a process for producinga split influenza virus or a subunit influenza preparation comprisingthe steps of: (i) providing a whole virus preparation; (ii) splittingthe whole virus preparation in the presence of a first detergent; (iii)adding t-octylphenoxypolyethoxyethanol (TRITON X-100™) to the resultingsplit virus preparation; and (iv) filtering the split virus preparation.

The invention also relates to a split influenza virus preparationsand/or subunit influenza virus preparations obtained or obtainable bysaid process.

The invention also relates to a process for preparing a pharmaceuticalor immunogenic composition, comprising the steps of: (i) providing asplit influenza virus or a subunit influenza preparation produced by aprocess of the invention as described herein, (ii) admixing said a splitinfluenza virus or a subunit influenza preparation with apharmaceutically acceptable carrier to prepare pharmaceutical orimmunogenic composition.

The invention also relates to a pharmaceutical or immunogeniccomposition obtained or obtainable by a process of the invention.

The invention also relates to a method of inducing an immune response ina human subject, said method comprising administering to the subjectsaid pharmaceutical or immunogenic composition as described herein.

The invention further relates to a pharmaceutical and/or immunogeniccomposition as defined herein for use in the treatment or prevention ofinfluenza disease or infection.

The invention also relates to an antigen preparation comprising aninfluenza virus hemagglutinin (HA) and a detergent wherein the weightratio of detergent (μg/ml) to hemagglutinin (μg/ml) is between 1.5 and15.

The invention also relates to a process for producing a split influenzavirus or subunit influenza preparation comprising the steps of: (i)providing a whole influenza virus preparation, (ii) splitting the wholevirus preparation in the presence of a detergent added in an amountsuitable to prevent aggregation of viral particles once split.

The invention also relates to a vaccine comprising one or more splitinfluenza virus preparations or subunit influenza preparations preparedaccording to any of the processes described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses various influenza vaccine production processes.

FIG. 2 discloses the loss of HA after several production steps withoutthe addition of t-octylphenoxypolyethoxyethanol (TRITON X-100™) 8/1:purified monovalent split virus bulk; 8/2 and 8/3: gradual filtration;8/4: Inactivation.

FIG. 3 discloses SDS-PAGE of H1N1v monovalent bulk produced with/withoutaddition of t-octylphenoxypolyethoxyethanol (TRITON X-100™).

FIG. 4 discloses HI titers (GMT+/−CI95) on Day 14 Post-II in BALB/c miceimmunised with the non-adjuvanted A/California/7/2009 split vaccineprepared without addition of t-octylphenoxypolyethoxyethanol (TRITONX-100™).

FIG. 5 discloses HI titers (GMT+/−CI95) on day 14 Post-II in BALB/c miceimmunized with the non-adjuvanted A/California/7/2009 split vaccineprepared with the addition of t-octylphenoxypolyethoxyethanol (TRITONX-100™).

FIG. 6 discloses HI titres (GMT+/−CI95) in BALB/c mice for AS03Aadjuvanted vaccine A/California/7/2009 NYMC X-179A without additionalt-octylphenoxypolyethoxyethanol (TRITON X-100™)-14PI.

FIG. 7 discloses inhibition of hemagglutination titres (GMT+/−CI95) inBALB/c mice for AS03A adjuvanted vaccine A/California/7/2009 NYMC X-179Awith additional t-octylphenoxypolyethoxyethanol (TRITON X-100™)-14PI.

FIG. 8 discloses inhibition of hemagglutination titres (GMT+/−CI95) inBALB/c mice for AS03A adjuvanted vaccine A/California/7/2009 NYMC X-179Awithout additional t-octylphenoxypolyethoxyethanol (TRITONX-100™)-14PII.

FIG. 9 discloses Inhibition of hemagglutination titres (GMT+/−CI95) inBALB/c mice for AS03A adjuvanted vaccine A/California/7/2009 NYMC X-179Awith additional t-octylphenoxypolyethoxyethanol (TRITON X-100™)-14PII.

DETAILED DESCRIPTION

The inventors have determined that the use oft-octylphenoxypolyethoxyethanol (TRITON X-100™) in combination with asplit virus preparation increases in the yield of antigenic componentsof the virus preparation. The process increases the yield of antigenfrom pandemic strains and seasonal (interpandemic) strains. Increasedyield is desirable, e.g. because it allows for the production of agreater number of vaccine doses at a time when such vaccine doses arecurrently insufficient.

The use of t-octylphenoxypolyethoxyethanol (TRITON X-100™) incombination with a split virus preparation or subunit preparation alsoimproves the purity of influenza antigens, e.g. HA or NA.

Without wishing to be bound by theory, the use oft-octylphenoxypolyethoxyethanol (TRITON X-100™) in combination with asplit influenza virus preparation helps prevent aggregation of the splitviral particles and allows the influenza vaccine to be more easilyfiltered, which accounts in whole or in part for the increased yieldobserved when using t-octylphenoxypolyethoxyethanol (TRITON X-100™).

The present invention relates generally to processes for producing asplit influenza virus or a subunit influenza preparation comprising thesteps of: (i) providing a whole virus preparation, (ii) splitting thewhole virus preparation in the presence of a first detergent, (iii)adding t-octylphenoxypolyethoxyethanol (TRITON X-100™) to the resultingsplit virus preparation; and (iv) filtering the split virus preparation

In another aspect, the present invention provides a process forproducing a split influenza virus preparation or a subunit influenzapreparation comprising the steps of: (i) providing a whole viruspreparation; (ii) splitting the whole virus preparation in the presenceof a first detergent and (iii) adding t-octylphenoxypolyethoxyethanol(TRITON X-100™) to the resulting split virus preparation; and (iv)filtering the split virus preparation, wherein the second detergent isused in step (iii) at a concentration of at least 0.1% (v/v).

The process may comprise a further step of inactivating said splitinfluenza virus preparation or subunit influenza preparation, suitablyafter step (iii).

Accordingly in a third aspect, the present invention provides a processfor producing a split influenza virus preparation or a subunit influenzapreparation comprising the steps of: (i) providing a whole viruspreparation, (ii) splitting the whole virus preparation in the presenceof a detergent; (iii) adding t-octylphenoxypolyethoxyethanol (TRITONX-100™) to the resulting split virus preparation; (iv) inactivating saidsplit virus preparation; and (v) filtering the split virus preparation

Detergents

Split influenza virus preparations have previously been produced using asolvent/detergent treatment, such as tri-n-butyl phosphate, ordiethylether in combination with polyoxyethylene sorbitan monooleate(TWEEN-80®, or POLYSORBATE 80™) (known as “Tween-ether” splitting) andthis process is still used in some production facilities. Othersplitting agents now employed include detergents or proteolytic enzymesor bile salts, for example sodium deoxycholate as described in patentno. DD 155 875 or in or in WO 02/097072 (U.S. Pat. No. 7,316,813B2),incorporated herein by reference. Detergents that can be used assplitting agents include cationic detergents e.g. cetyl trimethylammonium bromide (CTAB), other ionic detergents e.g. laurylsulfate,taurodeoxycholate, or non-ionic detergents such as the ones describedabove including t-octylphenoxypolyethoxyethanol (TRITON X-100™) (forexample in a process described in Lina et al, 2000, Biologicals 28,95-103) and Triton N-101, or combinations of any two or more detergents.

In one aspect the first detergent is selected from the group of: anionic detergent such as an anionic or cationic detergent, a non-ionicdetergent, a zwittergent or a combination thereof. Examples of suitabledetergents are sodium deoxycholate, CTAB andt-octylphenoxypolyethoxyethanol (TRITON X-100™).

The first detergent may also be used in combination with otherdetergents, such as those listed above, and may be a combination of nonionic detergents, for example a combination of sodium deoxycholate andpolyoxyethylene sorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™) orsodium deoxycholate and t-octylphenoxypolyethoxyethanol (TRITON X-100™).

In one aspect the first is not t-octylphenoxypolyethoxyethanol (TRITONX-100™). In a specific embodiment, residual amounts of the firstdetergent and t-octylphenoxypolyethoxyethanol (TRITON X-100™) arepresent in the final split influenza or subunit antigen preparation.

In a particular embodiment of the invention the first detergent ist-octylphenoxypolyethoxyethanol (TRITON X-100™).

In an alternative embodiment of the invention, the first detergent issodium deoxycholate.

Synonyms for t-octylphenoxypolyethoxyethanol (TRITON X-100™) include butare limited to polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenylether, octyl phenol ethoxylate, polyoxyethylene octyl phenyl ether,4-octylphenol polyethoxylate, Mono 30, TX-100, Octoxynol-9, octoxynol10, X-100, and octylphenol ethylene oxide condensate.

In one aspect t-octylphenoxypolyethoxyethanol (TRITON X-100™) is used incombination with other detergents, such as those listed above (forexample polyoxyethylene sorbitan monooleate (TWEEN-80™, or POLYSORBATE80™), such as an ionic detergent such as an anionic or cationicdetergent, a non-ionic detergent, a zwittergent or a combinationthereof.

The t-octylphenoxypolyethoxyethanol (TRITON X-100™) may be added beforeor during filtration of the split influenza preparation so as to improvefiltration. Accordingly, in one embodiment of the invention there isprovided a process for producing a split influenza virus or a subunitinfluenza preparation comprising the steps of: (i) providing a wholevirus preparation; (ii) splitting the whole virus preparation in thepresence of a first detergent; (iii) addingt-octylphenoxypolyethoxyethanol (TRITON X-100™) to the resulting splitvirus preparation; and (iv) filtering the split virus preparation,wherein steps (iii) and (iv) are performed simultaneously.

The t-octylphenoxypolyethoxyethanol (TRITON X-100™) added to splitinfluenza virus preparation is in an amount suitable to improve HA yieldcompared to a process without detergent treatment after splitting.Improved HA is suitably assessed in comparison to the results obtainedwithout detergent treatment after splitting, for example as measured byan SRD assay on [HA] after filtration through a 0.2 μm membrane (J. M.Wood et al.: An improved single radial immunodiffusion technique for theassay of influenza haemagglutinin antigen: adaptation for potencydetermination of inactivated whole virus and subunit vaccines. J. Biol.Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborativestudy of single radial diffusion and immunoelectrophoresis techniquesfor the assay of haemagglutinin antigen of influenza virus. J. Biol.Stand. 9 (1981) 317-330).

Accordingly, in one embodiment there is provided processes of theinvention wherein t-octylphenoxypolyethoxyethanol (TRITON X-100™) ispresent in an amount sufficient to improve HA yield in the filteredsplit virus preparation.

In a particular embodiment, there is provided processes of the inventionwherein the HA concentration in the filtered split influenza viruspreparation or subunit preparation is more than 50%, 75%, 100%, 150%,200% or 250% greater the HA compared to a process whereint-octylphenoxypolyethoxyethanol (TRITON X-100™) is not added to thesplit virus preparation prior to filtration.

In a particular embodiment t-octylphenoxypolyethoxyethanol (TRITONX-100™) is present in an amount greater than 0.025% (w/v). In aparticular embodiment t-octylphenoxypolyethoxyethanol (TRITON X-100™) ispresent in an amount of 0.1-1.5%. In further embodiments,t-octylphenoxypolyethoxyethanol (TRITON X-100™) is present in an amountof 0.1 to 0.8%. In yet a further embodiment,t-octylphenoxypolyethoxyethanol (TRITON X-100™) is present in an amountof 0.1 to 0.4%, e.g. 0.25% (w/v).

In one aspect the process of the invention comprises a filtration stepprior to an inactivation step, suitably substantially immediately priorto inactivation, and t-octylphenoxypolyethoxyethanol (TRITON X-100™) isadded before or during that filtration step.

The present invention thus relates in one aspect to a process forproducing a split or subunit influenza virus preparation comprising thesteps of: (i) providing a whole virus preparation, (ii) splitting thewhole virus preparation in the presence of a first detergent, (iii)adding t-octylphenoxypolyethoxyethanol (TRITON X-100™) to the resultingsplit virus preparation, (iv) filtering of the preparation, and (v)inactivating said filtered split virus preparation.

In an alternative embodiment of the invention there is provided aprocess for producing a split or subunit influenza virus preparationcomprising the steps of: (i) providing a whole virus preparation, (ii)splitting the whole virus preparation in the presence of a firstdetergent, (iii) adding t-octylphenoxypolyethoxyethanol (TRITON X-100™)to the resulting split virus preparation, and (iv) filtering theinactivated split virus preparation, further comprising the step ofinactivating the split virus preparation after step (iii) and beforestep (iv).

Suitably filtration is carried out through a filter ≤0.45 μm, such as0.2 μm or 0.22 μm. In one aspect the filtration is preceded by aprefiltration step. In one aspect the prefiltered product is sonicatedto facilitate the filtration step.

In one aspect t-octylphenoxypolyethoxyethanol is present during thesplitting process i.e. t-octylphenoxypolyethoxyethanol (TRITON X-100™)is present in addition to the first detergent.

In one aspect t-octylphenoxypolyethoxyethanol is present duringsplitting in an amount sufficient to prevent aggregation of viralparticles once split, suitably as determined by efficacy of filtrationof the split virus preparation, for example as determined by HAconcentration after filtration.

An alternative aspect of the invention comprises a process for producinga split influenza virus or subunit influenza preparation comprising thesteps of: (i) providing a whole virus preparation, (ii) splitting thewhole virus preparation in the presence oft-octylphenoxypolyethoxyethanol (TRITON X-100™) added in an amountsuitable to prevent loss of yield of HA after filtration, where loss ofyield is determined as compared to a process without such a detergentbeing present.

In this aspect t-octylphenoxypolyethoxyethanol (TRITON X-100™) ispresent during splitting to improve yield of HA. Use oft-octylphenoxypolyethoxyethanol (TRITON X-100™) after splitting may notbe required. The t-octylphenoxypolyethoxyethanol (TRITON X-100™) mayhelp in the splitting process, but this is not essential. Suitablyt-octylphenoxypolyethoxyethanol (TRITON X-100™) is able to help reducethe loss of HA that is manifested when the preparation is filtered.

Accordingly, in one embodiment there is provided processes of theinvention wherein steps (ii) and (iii) are performed simultaneously.

Influenza Strains

The whole, subunit or split influenza virus preparations as describedherein, may be derived from any influenza strain.

In one embodiment of the invention whole, subunit or split influenzavirus preparations as described herein is derived from an influenza A orinfluenza B strain. In one aspect influenza A virus strain is of a H1,H3, H7, H9 or H5 hemagglutinin sub-type. In one aspect the virus is apandemic or a potentially pandemic (e.g. H1N1v or H5N1) strain or a nonpandemic (inter-pandemic) (e.g. H3N2) strain. Data is provided hereinshowing the advantages of the present invention on both a pandemic(H1N1v such as A/California/7/2009 X-179A) and inter-pandemic H3N2strains.

Suitable strains from which the whole, subunit or split influenza viruspreparations of the invention are derived, include, but are not limitedto:

H1N1 Strains

A/New Caledonia/20/99-like strain, A/New Caledonia/20/99 (IVR-116),A/Solomon Islands/3/2006-like virus, A/Solomon Islands/3/2006 (IVR-145),A/Brisbane/59/2007-like virus, A/Brisbane/59/2007 IVR-148,A/Singapore/6/86-like, A/Singapore/6/86, A/Texas/36/91,A/Bayern/7/95-like, A/Johannesburg/82/96 (NIB-39),A/Beijing/262/95-like, A/Beijing/262/95 (X-127), A/NewCaledonia/20/99-like, A/New Caledonia/20/99 (IVR-116), A/SolomonIslands/3/2006-like, A/Solomon Islands/3/2006 (IVR-145),A/Brisbane/59/2007-like virus, A/Brisbane/59/2007 IVR-148

H3N2 Strains

A/Sydney/5/97-like strain, A/Sydney/5/97(IVR-108), A/Moscow/10/99-likestrain, A/Panama/2007/99 (RESVIR-17), A/Fujian/411/2002-like strain,A/Wyoming/3/2003 (X-147), A/Wellington/1/2004-like strain,A/Wellington/1/2004 (IVR-139), A/California/7/2004-like strain, A/NewYork/55/200 (NYMC X-157), A/Wisconsin/67/2005-like strain,A/Wisconsin/67/2005 (NYMC X-161-B), A/Brisbane/10/2007-like virus,A/Brisbane/10/2007 (IVR-147), A/Uruguay/716/2007 NYMC X-175C,A/Beijing/353/89-like, A/Guizhou/54/89, A/Beijing/353/89,A/Beijing/32/92, A/Shangdong/9/93, A/Johannesburg/33/94,A/Wuhan/359/95-like, A/Nanchang/933/95 (RESVIR-9), A/Sydney/5/97-like,A/Sydney/5/97 (IVR-108), A/Moscow/10/99-like, A/Panama/2007/99(RESVIR-17), A/Fujian/411/2002-like, A/Wyoming/3/2003 (X-147),A/Calefornia/7/2004-like, A/New York/55/2004 NYMC (X-157,A/Wisconsin/67/2005 (NYMC X-161), A/Wisconsin/67/2005-like,A/Wisconsin/67/2005 (NYMC X-161-B), A/Brisbane/10/2007-like virus,A/Uruguay/716/2007 NYMC X-175C.

B Strains

B/Beijing/184/93-like strain, B/Yamanashi/166/98, B/Sichuan/379/99-likestrain

B/Johannesburg/5/99, B/Sichuan/379/99-like strain, B/Johannesburg/5/99

B/Hong Kong/330/2001-like s., B/Shangdong/7/97, B/HongKong/330/2001-like s., B/Brisbane/32/2002, B/Shanghai/361/2002-likestrain, B/Jiangsu/10/2003, B/Malaysia/2506/2004-like strain,B/Malaysia/2506/2004, B/Florida/4/2006-like virus

B/Brisbane/3/2007, B/Yamagata/16/88, B/Panama/45/90, B/Harbin/7/94,B/Beijing/184/93-like, B/Beijing/184/93-like, B/Yamanashi/166/98,B/Sichuan/379/99-like, B/Johannesburg/5/99, B/Hong Kong/330/2001-like,B/Shangdong/7/97, B/Shanghai/361/2002-like, B/Jiangsu/10/2003,B/Malaysia/2506/2004-like, B/Malaysia/2506/2004, B/Florida/4/2006-likevirus, B/Brisbane/3/2007, B/Brisbane/60/2008-like virus,B/Brisbane/60/2008

Suitably the influenza virus strain or strains from which the whole,subunit or split influenza virus preparations are derived areinterpandemic (seasonal) strain(s), or strain(s) being associated with apandemic outbreak or having the potential to be associated with apandemic outbreak.

Interpandemic strains are for example strains which circulate globallyduring interpandemic periods such as but not limited to: H1N1, H1N2,H3N2 or B. Commercially available influenza vaccines are a trivalentcombination including one influenza B strain and two influenza A strains(H1N1, H3N2).

The features of an influenza virus strain that give it the potential tocause a pandemic or an outbreak of influenza disease associated withpandemic influenza strains are: it contains a new haemagglutinincompared to the haemagglutinin in the currently circulating strains andtherefore nearly all people are immunologically naive; it is capable ofbeing transmitted horizontally in the human population; and it ispathogenic for humans. A new haemagglutinin may be one which has notbeen evident in the human population for an extended period of time,probably a number of decades, such as H2. Or it may be a haemagglutininthat has not been circulating globally in the human population before,for example H5, H9, H7 or H6 which are found in avian species (birds).In either case the majority, or at least a large proportion of, or eventhe entire population has not previously encountered the antigen and/oris immunologically naïve to it. At present, the influenza A virus thathas been identified by the WHO as one that potentially could cause apandemic in humans is the highly pathogenic H5N1 avian influenza virus.Therefore, the pandemic vaccine disclosed herein suitably comprisesH5N1, H9N2 or H7N1.

The influenza virus strain may be a pandemic strain. Suitable pandemicstrains are, but not limited to: H5N1, H5N8, H5N9, H7N4, H9N2, H7N7,H7N3, H2N2 and H7N1. Other pandemic strains in human: H7N3, H10N7, H5N2and H7N2. An influenza strain which is a pandemic strain or a strainsusceptible to be associated with a pandemic will be referred to inshort in this document as a “pandemic strain”.

The whole, subunit or split influenza virus preparations as describedherein may be egg-derived or cell-culture derived. For example, whole,subunit or split influenza virus preparations according to the inventionmay be derived from the conventional embryonated egg method, by growinginfluenza virus in eggs and purifying the harvested allantoic fluid.Eggs can be accumulated in large numbers at short notice. Alternatively,they may be derived from any of the new generation methods using cell orcell culture to grow the virus or express recombinant influenza virussurface antigens. Suitable cell substrates for growing the virus includefor example dog kidney cells such as MDCK or cells from a clone of MDCK,MDCK-like cells, monkey kidney cells such as AGMK cells including Verocells, suitable pig cell lines, or any other mammalian cell typesuitable for the production of influenza virus for vaccine purposes.Suitable cell substrates also include human cells e.g. MRC-5 cells orthe Per.C6 cell line.

In a particular embodiment, whole, subunit or split influenza viruspreparations of the invention are derived from primary cells such aschicken embryo fibroblasts and avian cell lines such as chicken or duckcell lines (e.g. EBx cell line such as EB14 derived from chicken or EB24or EB66 derived from duck embryonic stem cells, disclosed in WO03/076601(US2004058441A1) and in WO08/129058 (US2010062489A1) are also included.Suitable insect cells are 519 or Hi5. In a particular embodiment thewhole, subunit or split influenza virus preparations of the inventionare derived from EB66 cells.

In one aspect the invention relates to a split influenza viruspreparation or subunit influenza preparation obtained or obtainable inthe process of the present invention.

In one aspect the invention relates to a process for preparing apharmaceutical composition, comprising the steps of: (i) providing asplit influenza virus preparation or subunit influenza preparation byany of the processes disclosed herein, and (ii) admixing said splitinfluenza virus preparation or subunit influenza preparation with apharmaceutically acceptable carrier to prepare the vaccine.

In one aspect the invention relates to a pharmaceutical and/orimmunogenic composition obtained or obtainable by any the processes ofthe invention.

A pharmaceutical composition of the invention containing a splitinfluenza virus preparation or subunit influenza preparation may bereferred to as an immunogenic composition or a vaccine herein.

Medical Treatment

In one aspect the invention relates to a method of inducing an immuneresponse into a human subject, said method comprising administering tothe subject the pharmaceutical and/or immunogenic composition or vaccineof the invention as described herein.

In a further embodiment of the invention there is provided apharmaceutical/immunogenic composition or vaccine as described hereinfor use in medicine.

In a further embodiment of the invention there is provided apharmaceutical/immunogenic composition or vaccine as described hereinfor use in the treatment and/or prevention of disease caused by theinfluenza virus in a subject.

In a further embodiment of the invention there is provided the use of apharmaceutical/immunogenic composition or vaccine as described herein inthe manufacture of a medicament for the treatment and/or prevention ofdisease caused by influenza virus in a subject.

In a particular embodiment of the invention, the subject administeredwith a pharmaceutical/immunogenic composition or vaccine as describedherein is immune-compromised. In a particular embodiment, the subject isover 60 years old, in particular 65 or more years old. In a furtherembodiment, the subject is an infant/child, in particular than 6 monthsold, and even more particular in particular between 6 and 23 months ofage.

Adjuvant

In one aspect a pharmaceutical or immunogenic composition of theinvention is not adjuvanted. In another aspect a pharmaceutical orimmunogenic composition of the invention comprises an adjuvant, forexample an oil in water emulsion.

In one aspect an adjuvant according to the present invention is anemulsion, in particular, an oil-in-water emulsion, and may optionallycomprise other immunostimulants.

In a specific embodiment, an oil-in-water emulsion comprises ametabolisable, non-toxic oil, such as squalane or squalene, optionally atocol such as tocopherol in particular alpha tocopherol (and optionallyboth squalene and alpha tocopherol) and an emulsifier (or surfactant)such as the non-ionic surfactant polyoxyethylene sorbitan monooleate(TWEEN-80™ or POLYSORBATE 80™). Mixtures of surfactants can be usedpolyoxyethylene sorbitan monooleate (TWEEN-80™ or POLYSORBATE80™)/sorbitan trioleate (SPAN 85™) mixtures, or polyoxyethylene sorbitanmonooleate (TWEEN-80™ or POLYSORBATE80™)/t-octylphenoxypolyethoxyethanol (TRITON X-100™) mixtures.

Tocols (e.g. vitamin E) are also used in oil emulsions adjuvants(EP0382271B1; U.S. Pat. No. 5,667,784; WO95/17210). Tocols used in oilemulsions (optionally oil-in-water emulsions) may be formulated asdescribed in U.S. Pat. Nos. 5,650,155A; 5,667,784A; EP0382271B1, in thatthe tocols may be dispersions of tocol droplets, optionally comprisingan emulsifier, of optionally less than 1 micron in diameter.Alternatively, the tocols may be used in combination with another oil,to form the oil phase of an oil emulsion. Examples of oil emulsionswhich may be used in combination with the tocol are described herein,such as the metabolisable oils described above.

In an oil-in-water emulsion, the oil and emulsifier should be in anaqueous carrier. The aqueous carrier may be, for example, phosphatebuffered saline or a citrate buffer.

In one aspect, the oil in water emulsion has one of the followingcompositions:

-   -   from 0.5 to 11 mg squalene, from 0.05 to 5% polyoxyethylene        sorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™) and        optionally, from 2 to 12% alpha-tocopherol; or    -   about 5% squalene, about 0.5% polyoxyethylene sorbitan        monooleate (TWEEN-80™ or POLYSORBATE 80™) and about 0.5%        sorbitan trioleate (SPAN 85™). This adjuvant is called MF59.

Pharmaceutical and/or Immunogenic Compositions

In one embodiment pharmaceutical and/or immunogenic compositions of theinvention comprise hemagglutinin of one or more strains at an amount ofabout 15 μg/strain, about 7.5 μg/strain, about 3.8 μg/strain, about 1.9μg/strain or 5 μg/strain.

Some currently existing vaccines have residual detergent present as aresult of the use of that detergent in the splitting process. In othercases there may be addition of the detergent to the vaccine antigen. Inthe present invention the use of a detergent, such as a non-ionicsurfactant, such as t-octylphenoxypolyethoxyethanol (TRITON X-100™),added after splitting of the virus (and suitably prior to filtration andsuitably before inactivation), provides a ratio of between 1.5 and 15t-octylphenoxypolyethoxyethanol (TRITON X-100™): HA in the finalmonovalent bulk produced after the manufacturing process, assessed bythe weight/volume ratios of the t-octylphenoxypolyethoxyethanol (TRITONX-100™) and HA, suitably between 2.5 and 15, suitably between 3 and 15,suitably between 3.3 and 15, or a higher ratio.

In one aspect the invention relates to a pharmaceutical or immunogeniccomposition comprising an influenza virus hemagglutinin (HA) and a nonionic surfactant wherein the weight/volume ratio of non-ionic surfactantto hemagglutinin is between 1 and 15. The surfactant may bet-octylphenoxypolyethoxyethanol (TRITON X-100™). The HA concentrationmay be between 2 and 200 μg per strain per ml. Thet-octylphenoxypolyethoxyethanol (TRITON X-100™) concentration may bebetween 10 and 500 μg/ml.

In one aspect the invention relates to a vaccine made from multipleinfluenza strains, wherein at least one component is manufacturedaccording the process of the invention. Suitably a vaccine comprising 2or 3 strains are made using a process of the invention.

In one aspect pharmaceutical or immunogenic composition of the inventioncomprising multivalent strains, have a ratio of between 1 and 15t-octylphenoxypolyethoxyethanol (TRITON X-100™): HA in the finalvaccine, assessed by the weight/volume ratios of thet-octylphenoxypolyethoxyethanol (TRITON X-100™) and HA, suitably, suchas between 1.5 and 15, such as between 2 and 15, such as between 2.5 and15, suitably between 3 and 15, suitably between 3.3 and 15, or a higherratio.

Split Influenza Preparation

The preparation process for a split influenza virus preparation for usein pharmaceutical or immunogenic compositions may include a number ofdifferent filtration and/or other separation steps such asultracentrifugation, ultrafiltration, zonal centrifugation andchromatography (e.g. ion exchange) steps in a variety of combinations,and optionally an inactivation step e.g. with heat, formaldehyde orβ-propiolactone or U.V. which may be carried out before or aftersplitting. The splitting process may be carried out as a batch,continuous or semi-continuous process. A preferred splitting andpurification process for a split immunogenic composition is described inWO 02/097072 (U.S. Pat. No. 7,316,813B2) which is incorporated byreference in its entirety.

Such a process suitably comprises the steps of:

Initial filtration, virus splitting, filtration, inactivation,filtration; wherein the filtration may be an ultrafiltration step.

Accordingly, in one embodiment there is provided processes of theinvention further comprising the step of inactivating the splitinfluenza virus preparation. Inactivation can be performed by any methodknown to the skilled person including, but is not limited to usingformaldehyde. Inactivation may be performed at any stage of the processfollowing step (ii). In a particular embodiment of the inventioninactivation is performed after step (iii).

The process of the invention comprise at least one filtration step (stepiv), but may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or more filtration stepsin addition to that of step (iv). For example, the split influenza viruspreparation may be filtered using a series of filter of differing sizee.g. 0.5 μm, 0.45 μm and/or 0.2/0.22 μm filters. In a particularembodiment the filtering step (step iv) is performed using one or morefilter membranes, wherein at least one filter membrane is sterile grade(e.g. 0.2 μm or 0.22 μm). In a further embodiment there is providedprocesses of the invention comprising the step of filtering the splitinfluenza virus preparation in addition to the filtration step of step(iv). In a particular embodiment, the filtering step in addition to thatof step iv) is performed using a sterile grade filter (for example a 0.2μm or 0.22 μm filter).

In a further embodiment, there are provided processes of the inventionfurther comprising the step of ultracentrifuging the split influenzavirus preparation. Suitably, ultrafiltration is performed using acellulose acetate membrane with about 20 kDa MW cut-off

In a further embodiment, there are provided processes of the inventionfurther comprising the step of clarifying the whole virus preparation.

In a further embodiment, there are provided processes of the inventioncomprising the step of ultracentrifuging the whole virus preparation.

Preferred split influenza virus preparations according to the inventioncomprise a residual amount of polyoxyethylene sorbitan monooleate(TWEEN-80™ or POLYSORBATE 80™) and/or t-octylphenoxypolyethoxyethanol(TRITON X-100™) remaining from the production process, although thesemay be added or their concentrations adjusted after preparation of thesplit antigen. In one embodiment both polyoxyethylene sorbitanmonooleate (TWEEN-80™ or POLYSORBATE 80™) andt-octylphenoxypolyethoxyethanol (TRITON X-100™) are present. Thepreferred ranges for the final concentrations of polyoxyethylenesorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™) in the vaccine dose,arising from the antigenic preparation, are:

polyoxyethylene sorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™): 0.01to 1%, or about 0.1% (v/v)

In a specific embodiment, the final concentration for polyoxyethylenesorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™) arising from thesplit influenza virus preparation, ranges from 0.025%-0.09% w/v. Inanother specific embodiment, the split influenza virus preparation isprovided as a 2 fold concentrated mixture, which has a polyoxyethylenesorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™) concentration rangingfrom 0.025%-0.2% (w/v) and has to be diluted two times upon finalformulation with the adjuvanted (or the buffer in the controlformulation).

In one embodiment, the split influenza virus preparation is prepared inthe presence of low level of thiomersal, or in the absence ofthiomersal. In another embodiment, the resulting split influenza viruspreparation is stable in the absence of organomercurial preservatives,in particular the preparation contains no residual thiomersal. Inparticular the split influenza virus preparation comprises ahaemagglutinin antigen stabilised in the absence of thiomersal, or atlow levels of thiomersal (generally 5 μg/ml or less). Specifically thestabilization of B influenza strain is performed by a derivative ofalpha tocopherol, such as alpha tocopherol succinate (also known asvitamin E succinate, i.e. VES). Such preparations and methods to preparethem are disclosed in WO 02/097072 (U.S. Pat. No. 7,316,813B2) which isincorporated by reference in its entirety.

A preferred composition contains three inactivated split influenza viruspreparation prepared from the WHO recommended strains of the appropriateinfluenza season.

In one embodiment, the split influenza virus preparation or subunitinfluenza preparation and the adjuvant according to the invention arecontained in the same container. It is referred to as ‘one vialapproach’. In another embodiment, the vial is a pre-filled syringe. Inan alternative embodiment, the split influenza virus preparation orsubunit influenza preparation and adjuvant according to the inventionare contained in separate containers or vials and admixed shortly beforeor upon administration into the subject. It is referred to as ‘two vialsapproach’. Suitably the two vials approach consists of 0.5 ml ofconcentrated split influenza virus preparation or subunit influenzapreparations as described herein presented in a type I glass vial(antigen container) and of a pre-filled type I glass syringe containing0.5 ml of the adjuvant (adjuvant container). Alternatively the two vialapproach is presented in 2 vials (one for the antigen one for theadjuvant, of 10 doses each) for mixture prior to the administration tothe first patient within 24 hours at room temperature and subsequentstorage at 4° C. for a short period of time (e.g. up to one week) forsubsequent administration. At the time of injection, the content of themulti-dose vial or the syringe containing the adjuvant is injected intothe vial that contains the concentrated split influenza viruspreparation or subunit influenza preparation. After mixing the contentis withdrawn into the syringe and the needle is replaced by anintramuscular needle. One dose of the adjuvanted split influenza viruspreparation or subunit influenza preparation (pharmaceutical orimmunogenic composition) corresponds to 0.5 ml.

In one embodiment, each human dose of the pharmaceutical or immunogeniccomposition contains a 15 μg of HA per influenza strain per dose, asdetermined by SRID. This is particularly useful for the elderlypopulation.

An important aspect of the present invention is the fact that theinfluenza antigen(s) can be used at lower amounts than had previouslybeen thought useful, suitably at a level of less than 15 μg HA perstrain of virus, for example between 1 and 10 μg HA per strain, perhuman dose of the immunogenic composition.

Accordingly, in one embodiment, each human dose of the pharmaceutical orimmunogenic composition contains a low dose of haemagglutinin (HA),defined as an amount of less than 15 μg of HA per dose, suitably lessthan 10 μg, as measured by single radial immunodiffusion (SRD) (J. M.Wood et al.: J. Biol. Stand. 5 (1977) 237-247; J. M. Wood et al., J.Biol. Stand. 9 (1981) 317-330). In a specific embodiment, the human doseof the immunogenic composition comprises a dose of haemagglutinin (HA)per strain at a level of about 10 μg, for example between 5 and 15 μg,suitably between 6 and 14 μg, for example between 7 and 13 μg or between8 and 12 μg or between 9 and 11 μg, or 10 μg. In a further embodiment,the human dose of the immunogenic composition comprises a dose ofhaemagglutinin (HA) per strain at a level of about 5 μg, for examplebetween 1 and 9 μg, or between 2 and 8 μg or suitably between 3 and 7 μgor 4 and 6 μg, or 5 μg. Suitable amounts are 1.9 μg, 2.5 μg, 3.8 μg, 5.0μs, 7.5 μs, or 10 μg HA or any suitable amount of HA lower than 15 μgwhich would have be determined such that the vaccine composition meetsthe efficacy criteria as defined herein. Advantageously an HA dose of 1μg of HA or even less such as 0.5 μg of HA that would allow meeting theregulatory criteria defined in Tables C or/and D may be used. A suitableamount of HA is for example any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, μg (w/v) per influenza strain per human dose of theimmunogenic composition. Said low amount of HA may be as low aspractically feasible provided that it allows to formulate a vaccinewhich meets the international e.g. EU or FDA criteria for efficacy, asdetailed below (see Table 3 and 4 and the specific parameters as setforth).

A pharmaceutical or immunogenic composition dose of 0.5 ml is suitablyused. A pharmaceutical or immunogenic composition dose of 1 ml (0.5 mladjuvant plus 0.5 ml antigen preparation) is also suitable.Advantageously, a pharmaceutical or immunogenic composition doseaccording to the invention, in particular a low HA amount vaccine, maybe provided in a smaller volume than the conventional injected split fluvaccines, which are generally about 0.5, 0.7 or 1 ml per dose. The lowvolume doses according to the invention are suitably below 500 μl,typically below 300 μl and suitably not more than about 200 μl or lessper dose. Slight adaptation of the dose volume will be made routinelydepending on the HA concentration in the original bulk sample, ordepending on the delivery route with smaller doses being given by theintranasal or intradermal route, or depending on the target population(for example infants may receive half of an adult human dose).

The influenza pharmaceutical or immunogenic compositions of theinvention suitably meet certain international criteria for vaccines.Standards are applied internationally to measure the efficacy ofinfluenza vaccines. Serological variables are assessed according tocriteria of the European Agency for the Evaluation of Medicinal Productsfor human use (CHMP/BWP/214/96, Committee for Proprietary MedicinalProducts (CPMP). Note for harmonization of requirements for influenzavaccines, 1997. CHMP/BWP/214/96 circular No 96-0666:1-22) for clinicaltrials related to annual licensing procedures of influenza vaccines(Table 3 or Table 4). The requirements are different for adultpopulations (18-60 years) and elderly populations (>60 years) (Table C).For interpandemic influenza vaccines, at least one of the assessments(seroconversion factor, seroconversion rate, seroprotection rate) shouldmeet the European requirements, for all strains of influenza included inthe vaccine. The proportion of titres equal or greater than 1:40 isregarded most relevant because these titres are expected to be the bestcorrelate of protection [Beyer W et al. 1998. Clin Drug Invest.;15:1-12].

As specified in the “Guideline on dossier structure and content forpandemic influenza vaccine marketing authorisation application.(CHMP/VEG/4717/03, Apr. 5, 2004, or more recentlyEMEA/CHMP/VWP/263499/2006 of 24 Jan. 2007 entitled ‘Guidelines on fluvaccines prepared from viruses with a potential to cause a pandemic’,available on www.emea.eu.int), in the absence of specific criteria forinfluenza vaccines derived from non circulating strains, it isanticipated that a pandemic candidate vaccine should (at least) be ableto elicit sufficient immunological responses to meet suitably all threeof the current standards set for existing vaccines in unprimed adults orelderly subjects, after two doses of vaccine. The EMEA Guidelinedescribes the situation that in case of a pandemic the population willbe immunologically naive and therefore it is assumed that all three CHMPcriteria for seasonal vaccines will be fulfilled by pandemic candidatevaccines. No explicit requirement to prove it in pre-vaccinationseronegative subjects is required.

The compositions of the present invention suitably meet at least onesuch criteria for the strain included in the composition (one criteriais enough to obtain approval), suitably at least two, or typically atleast all three criteria for protection as set forth in Table 3.

TABLE 3 (CHMP criteria) 18-60 years >60 years Seroconversionrate* >40% >30% Conversion factor** >2.5 >2.0 Protectionrate*** >70% >60% *Seroconversion rate is defined as the proportion ofsubjects in each group having a protective post-vaccination titer ≥1:40.The seroconversion rate simply put is the % of subjects who have an HItiter before vaccination of <1:10 and ≥1:40 after vaccination. However,if the initial titer is ≥1:10 then there needs to be at least a fourfoldincrease in the amount of antibody after vaccination. **Conversionfactor is defined as the fold increase in serum HI geometric mean titers(GMTs) after vaccination, for each vaccine strain. ***Protection rate isdefined as the proportion of subjects who were either seronegative priorto vaccination and have a (protective) post-vaccination HI titer of≥1:40 or who were seropositive prior to vaccination and have asignificant 4-fold increase in titer post-vaccination; it is normallyaccepted as indicating protection.

FDA uses slightly different age cut-off points, but their criteria arebased on the CHMP criteria. Appropriate endpoints similarly include: 1)the percent of subjects achieving an HI antibody titer ≥1:40, and 2)rates of seroconversion, defined as a four-fold rise in HI antibodytiter post-vaccination. The geometric mean titer (GMT) should beincluded in the results, but the data should include not only the pointestimate, but also the lower bound of the 95% confidence interval of theincidence rate of seroconversion, and the day 42 incidence rate of HItiters ≥1:40 must exceed the target value. These data and the 95%confidence intervals (CI) of the point estimates of these evaluationsshould therefore be provided. FDA draft guidance requires that bothtargets be met. This is summarised in Table 4.

TABLE 4 18-64 years >64 years Seroconversion rate* >40% >30% Rate of HItiters ≥1:40 >70% >60% *The seroconversion rate is defined as: a) forsubjects with a baseline titer ≥1:10, a 4-fold or greater rise; or b)for subjects with a baseline titer <1:10, a rise to ≥1:40. Thesecriteria must be met at the lower bound of the 95% CI for the truevalue.

In an alternative embodiment, the compositions of the present inventionsuitably meet at least one such criteria for the strain included in thecomposition, suitably both criteria for protection as set forth in Table4.

Suitably this effect is achieved with a low dose of antigen, such aswith 7.5 μg HA or even a lower antigen dose such as 3.8 μg or 1.9 μg ofHA.

Suitably any or all of such criteria are also met for other populations,such as in children and in any immuno-compromised population.

In one aspect of the invention, the human dose of the pharmaceutical orimmunogenic composition contains an haemagglutinin (HA) from a singleinfluenza strain, and is referred to as a “monovalent” influenzacomposition. In another aspect of the invention, the human dose of thepharmaceutical or immunogenic composition comprises haemagglutinin (HA)from more than one influenza strain, and is referred to as a“multivalent” influenza composition. A suitable multivalent compositionaccording to the invention is a bivalent composition (comprisinghaemagglutinin (HA) from two influenza virus strains such as but notexclusively two strains associated to a pandemics or susceptible to beassociated with a pandemic, e.g. H5=H2), a trivalent composition(comprising haemagglutinin (HA) from three influenza virus strains,optionally from two A strains, and one B strain such as but not limitedto B/yamagata or B/Victoria), a quadrivalent composition (comprisinghaemagglutinin (HA) from four influenza virus strains) or a pentavalentcomposition (comprising haemagglutinin (HA) from five influenza virusstrains). A suitable quadrivalent composition comprises haemagglutininfrom two A strains and two B strains from different lineage (such asB/yamagata or B/Victoria). Such a composition comprising a second Bstrain is particularly suitable for very young children especially whereprior exposure or priming is important. Alternatively a quadrivalentcomposition comprises haemagglutinin from three A strains (optionallyH1N1, H3N2, and one A strain associated to a pandemic or susceptible tobe associated to a pandemic) and one B strain (such as B/yamagata orB/Victoria). Another alternative quadrivalent composition compriseshaemagglutinin from four A strains from a strain associated to apandemic or susceptible to be associated to a pandemic, such as avianstrains such as H5+H2+H7+H9. Specifically a multivalent adjuvantedpandemic composition such as a pandemic bi-valent (e.g. H5+H2) ortrivalent or quadrivalent (e.g. H5+H2+H7+H9) offers the advantage of apre-emptive immunisation against pandemic influenza A threats subtypesand durable priming against threat subtypes. Typically two doses aregiven from 6 weeks of age using a convenient schedule (e.g., 6-12 monthsapart), and optionally a periodic booster foreseen (e.g., 10 yrs).Optionally, such a pandemic vaccine may be combined with a seasonalvaccine.

A multivalent composition can also comprise more than 5 influenzastrains such as 6, 7, 8, 9 or 10 influenza strains.

When two B strains are used in a multivalent seasonal composition, theycan be from two different lineages (optionally from B/Victoria andB/Yamagata). At least one of said B strain, suitably both B strains,will be from a circulating lineage. Such a composition is particularlysuitable for children. Suitably when the multivalent composition for usein children includes two B strains the quantity of antigen normallyallotted to the B strain is divided among the two B strains.Specifically, the adjuvanted quadrivalent (H1+H3+ both B lineages)influenza vaccine offers the advantage of enhanced prophylaxis for naïvechildren as its superior efficacy compared to unadjuvanted vaccines (interms of both homologous and drift protection, and its efficacy againsttwo circulating B lineages) and of possible year-round immunizationbased on age. One dose or two doses are suitably administered as earlyas from the age of 6 weeks, or between 6 to 35 months.

In a specific embodiment, the human dose of the pharmaceutical orimmunogenic composition is a trivalent immunogenic or vaccinecomposition comprising haemagglutinin (HA) from two A strains(optionally H1N1, H3N2) and one B strain. Suitably the HA per strain isa low amount of HA (optionally 10 μg HA per strain or below) and is asdefined above. Suitably the HA per strain is at about or below 5 μg, atabout 2.5 μg or below. An adjuvant as defined herein may be included andin particular as defined in Table 1. Suitably the adjuvant compositionis an oil-in-water emulsion comprising squalene, alpha-tocopherol, andpolyoxyethylene sorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™) at anamount of between 5-6 mg, between 5-6 mg and between 2-3 mg per dose,respectively. Alternatively, the adjuvant composition is an oil-in-wateremulsion comprising squalene, alpha-tocopherol, and polyoxyethylenesorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™) at an amount ofbetween 2.5-3.5 mg, between 2-3 mg and between 1-2 mg per dose,respectively. These adjuvanted immunogenic compositions or vaccines areparticularly suitable for the adult (18-60 years) or older children(3-17 years) population, and may provide cross-protection against H3N2drift variants and against B strain from a different lineage.

In another specific embodiment, the human dose of the immunogeniccomposition is a quadrivalent immunogenic or vaccine compositioncomprising haemagglutinin (HA) from two A strains (optionally H1N1,H3N2) and two B strains (optionally from a different lineage, such asfrom B/Victoria and B/Yamagata). In another specific embodiment, thehuman dose of the immunogenic composition is a quadrivalent immunogenicor vaccine composition comprising haemagglutinin (HA) from twointerpandemic A strains (optionally H1N1, H3N2), one B strain and one Astrain associated to a pandemic or susceptible to be associated with apandemic (optionally H5N1, H9N2, H7N7, H5N8, H5N9, H7N4, H7N3, H2N2,H10N7, H5N2, H7N2 and H7N1). In another specific embodiment, the humandose of the immunogenic composition is a quadrivalent immunogenic orvaccine composition comprising haemagglutinin (HA) from threeinterpandemic A strains (optionally H1N1, and two H3N2 strains) and oneB strain. Suitably the HA per strain per dose is at about 15 μg.Suitably the HA per strain is a low amount of HA (optionally at about 10μg HA per strain per dose or below, so as to achieve a maximum of 40-45μg HA per dose of quadrivalent composition) and is as defined above.Suitably the HA per strain is at about or below 5 μg, at about 2.5 orbelow. An adjuvant where present may be any adjuvant as describedherein.

In another specific embodiment, the human dose of the pharmaceutical orimmunogenic composition is a pentavalent immunogenic or vaccinecomposition comprising haemagglutinin (HA) from two interpandemic Astrains (optionally H1N1, H3N2), two B strains (optionally from adifferent lineage, such as from B/Victoria and B/Yamagata) and one Astrain associated to a pandemic or susceptible to be associated with apandemic (optionally H5N1, H9N2, H5N8, H5N9, H7N4, H7N7, H7N3, H2N2,H10N7, H5N2 and H7N1). In another specific embodiment, the human dose ofthe pharmaceutical or immunogenic composition is a pentavalentimmunogenic or vaccine composition comprising haemagglutinin (HA) fromthree interpandemic A strains (optionally H1N1, and two H3N2 strains)and two B strains (optionally from a different lineage, such as fromB/Victoria and B/Yamagata). Suitably the HA per strain is a low amountof HA (optionally 10 μg HA per strain or below) and is as defined above.

In one embodiment the multivalent compositions are adjuvanted, suitablywith a squalene-based oil-in-water emulsion adjuvant. Accordingly in aspecific embodiment the invention provides an influenza immunogeniccomposition comprising squalene and HA wherein the weight ratiosqualene:total amount of HA (all influenza strains included) is in therange of between about 50-150 or about 150-400 (e.g. about 200-300).Such compositions are suitably but not exclusively for use in theelderly population and best balances reactogenicity and immunogenicity.In another embodiment, the invention provides an influenza immunogeniccomposition comprising squalene and HA wherein the weight ratiosqualene:total amount of HA (all influenza strains included) is betweenabout 50-400, e.g. about 50-100, 75-150, 75-200, 75-400, 100-200,100-250 or 200-400. The ratio will suitably be such that at least two,suitably all three criteria (Table C or D) for protection will be metfor a specific population. Suitably the HA is from at least three, atleast four influenza strains. Suitably three seasonale (e.g. H1N1, H3N2,B) strains are present. Suitably when four strains are present they arefrom the group of: four seasonal strains (e.g. H1N1, H3N2, two Bstrains; or H1N1, B, two H3N2 strains) or the group of one pandemic(e.g. avian) strain plus three seasonal strains (e.g. H1N1, H3N2, B).

General Language

The teaching of all references in the present application, includingpatent applications and granted patents, are herein fully incorporatedby reference. Any patent application to which this application claimspriority is incorporated by reference herein in its entirety in themanner described herein for publications and references.

For the avoidance of doubt the terms ‘comprising’, ‘comprise’ and‘comprises’ herein is intended by the inventors to be optionallysubstitutable with the terms ‘consisting of’, ‘consist of’, and‘consists of’, respectively, in every instance. Embodiments hereinrelating to “vaccine compositions” of the invention are also applicableto embodiments relating to “immunogenic compositions” of the invention,and vice versa. The term “about” (or “around”) in all numerical valuesallows for a 5% variation, i.e. a value of about 1.25% would mean frombetween 1.19%-1.31%.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for themeasurement, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The invention will be further described by reference to the following,non-limiting, examples:

EXAMPLES Example 1

An exemplary production process for the influenza virus strains based onthe process described in WO02/097072 (U.S. Pat. No. 7,316,813B2) and thesteps summarized in FIG. 1 was compared to two other processes asoutlined in FIG. 1.

The pandemic H1N1v strain (A/California/7/2009 X-179A) was producedaccording to Process A performed in the presence or the absence oft-octylphenoxypolyethoxyethanol (TRITON X-100™) (FIG. 1). When applyingthe manufacturing process A it was observed that approximately 50% ofhaemagglutinin (HA) as measured by HPLC was lost at the two 0.2 μmfiltration steps (FIG. 2).

Three test lots of A/California/7/2009 X-179A H1N1v monovalent bulkswere produced using the manufacturing process A amended by the additionof t-octylphenoxypolyethoxyethanol (TRITON X-100™) in the dilutionbuffer added after the splitting gradient, just before inactivation withformaldehyde (described herein as “process A+t-octylphenoxypolyethoxyethanol (TRITON X-100™)”).

Process steps upstream of the addition oft-octylphenoxypolyethoxyethanol (TRITON X-100™) were unchanged.

As shown in Table 5, the addition of t-octylphenoxypolyethoxyethanol(TRITON X-100™) after splitting and before filtration produced animproved yield of HA in comparison to the process withoutt-octylphenoxypolyethoxyethanol (TRITON X-100™) at this stage.

The particle sizes prior to filtration were found to be reduced comparedthose seen with process A which does not comprise the addition oft-octylphenoxypolyethoxyethanol (TRITON X-100™).

The HA yield at the monovalent bulk step is of 39 μg/egg as compared to15 μg/egg in process A +t-octylphenoxypolyethoxyethanol (TRITON X-100™),which corresponds to a 2.6-fold increase.

The ratio of t-octylphenoxypolyethoxyethanol (TRITON X-100™) content/HAcontent in H1N1v final bulk is higher when using process A+t-octylphenoxypolyethoxyethanol (TRITON X-100™), as compared to theprocess A alone without t-octylphenoxypolyethoxyethanol (TRITON X-100™).

The modified purification process (process A+t-octylphenoxypolyethoxyethanol (TRITON X-100™)) was also carried outon an H3N2 strain with the addition of t-octylphenoxypolyethoxyethanol(TRITON X-100™) at the same point in the process as used for the H1N1vstrain. The addition of t-octylphenoxypolyethoxyethanol (TRITON X-100™)in a dilution buffer after the splitting gradient, just beforeinactivation with formaldehyde, resulted in a 2-3 fold increase in theA/H2N2/Nanchang/933/95 RESVIR-9 strain.

TABLE 5 A/H1N1v strain H3N2 strain Process A with t- Fluarix processwith t- octylphenoxypolyethoxyethanol octylphenoxypolyethoxyethanolProcess A (TRITON X-100 ™) buffer (TRITON X-100 ™) HA yield per egg 15μg/egg

39 μg/egg 63 μg/egg HA content monovalent bulks 20-50 μg/ml 140-190μg/ml 80-280 μg/ml HA content final bulks 15 μg/ml 15 μg/ml 90 μg/ml t-0 μg/ml 470-600 μg/ml 190-440 μg/ml octylphenoxypolyethoxyethanol(TRITON X-100 ™) content monovalent bulks t- 13-17 μg/ml 60-103 μg/ml80-105 μg/ml octylphenoxypolyethoxyethanol (TRITON X-100 ™) contentfinal bulks t- 11-20 μg/ml 50-170 μg/ml 50-170 μg/mloctylphenoxypolyethoxyethanol (TRITON X-100 ™) Specification in finalbulk Ratio t- 0.7-1.3 3.3-11.3 0.6-1.9 octylphenoxypolyethoxyethanol(TRITON X-100 ™)/HA

Example 2: Impact on the Quality of the Antigen

The potential impact of t-octylphenoxypolyethoxyethanol (TRITON X-100™)on the quality of the product was evaluated by particle sizemeasurements, as well as purity and protein pattern analysis bySDS-PAGE.

Profile analysis by Gel and Western blot.

The addition of t-octylphenoxypolyethoxyethanol (TRITON X-100™) in theprocess is shown to improve purity, as the ratio of total protein/HAcontent is shown to be reduced from 2.5 to 1.7.

As seen in FIG. 3, the pattern of peptide bands in SDS-PAGE gels isunchanged whether t-octylphenoxypolyethoxyethanol (TRITON X-100™) wasused or not in the manufacturing process. An increased portion of HA(approximately 3-fold seen by densitometry) is observed in lots producedwith t-octylphenoxypolyethoxyethanol (TRITON X-100™). This finding isconsistent with the improved protein/HA ratio.

Example 3: Preliminary Quality Data on the 1st Pandemic Monovalent BulksLots Manufactured with t-Octylphenoxypolyethoxyethanol (TRITON X-100™)

Preliminary quality data obtained on three monovalent bulks producedusing the t-octylphenoxypolyethoxyethanol (TRITON X-100™) process arepresented in Table 6.

The data obtained are in line with what was seen on test lots producedfor the 1^(st) evaluations of the process: higher HA yields and higherpurity profile.

Batch analysis data on H1N1v monobulks manufactured with thet-octylphenoxypolyethoxyethanol (TRITON X-100™) process

A/California/7/2009 NYMC X-179A Lot N^(o) DFLSIDA030 DFLSIDA031DFLSIDA032 Parameter (AFLSFDA107) (AFLSFDA109) (AFLSFDA111) HA contentSRD (μg/ml) ~180 >=200 >=200 Protein (μg/mL) 259 297 325 Tween (μg/mL)503 ND ND t-octylphenoxypolyethoxyethanol 509 447 579 (TRITON X-100 ™)(μg/ml) Ratio t- ~2.83 ~2.24 ~2.88 octylphenoxypolyethoxyethanol (TRITONX-100 ™):HA

As a comparison, data obtained on H3N2 monovalent bulks manufactured foran H3N2 Fluarix vaccine made using t-octylphenoxypolyethoxyethanol(TRITON X-100™), as disclosed above are presented in Table 7.

TABLE 7 Batch analysis data obtained on H3N2 monovalent bulksA/Uruguay/716/2007 NYMC X-175C Lot N^(o) Parameter AFLUFDA070 AFLUFDA071AFLUFDA072 HA content SRD Int (μg/ml) 69 82 90 Sucrose concentration(μg/mL) 80 89 88 Protein (μg/mL) 172 232 198 Ovalbumin (ng/mL) 9 12 19Sodium Deoxycholate (μg/mL) 95 105 125 Endotoxin (EU/mL) <0.5 <0.5 <0.5Formaldehyde (μg/mL) 0.9 0.9 0.4 Tween (μg/mL) 391 438 407t-octylphenoxypolyethoxyethanol 374 431 474 (TRITON X-100 ™) (μg/ml)Viral inactivation Proved Proved Proved Sterility (according to Ph.Eur.)sterile sterile sterile Ratio t-octylphenoxypolyethoxyethanol 5.42 5.265.27 (TRITON X-100 ™):HA

Stability data obtained on representative H3N2 monovalent bulksmanufactured using the modified seasonal Fluarix process containingt-ctylphenoxypolyethoxyethanol (TRITON X-100™) are presented in Table 9and Table 10. The ratio of t-octylphenoxypolyethoxyethanol (TRITONX-100™)/HA contents pertaining to the lots followed for up to 12 monthsat 2-8° C. are all close to 3 (see Table 8), and the HA contents areshown to be stable overtime, with comparable values for each time pointgenerated and each lot tested.

Where necessary, t-octylphenoxypolyethoxyethanol (TRITON X-100™) can bedetected in the parts per million range by spectrophotometricmeasurement of the concentration of a t-octylphenoxypolyethoxyethanol(TRITON X-100™)-ammonium-cobalt-thiocyanate complex, seet-octylphenoxypolyethoxyethanol (TRITON X-100™) product information fromSigma and Crabb, N. T. And Persinger, H. E., J. Amer. Oil Chem. Soc.,41, 752-755 (1964) and Greff, R. A. et al., J. Amer. Oil Chem. Soc., 42,180-185 (1965).

TABLE 8 Ratio t-octylphenoxypolyethoxyethanol (TRITON X-100 ™)/HAcontents on the monovalent bulk lots of A/Uruguay/716/2007 NYMC X-175C(H3N2) t- Ratio t- octylphenoxy- octylphenoxy- polyethoxyethanolpolyethoxyethanol HA content (TRITON (TRITON Lot (μg/ml) Int X-100 ™)(μg/ml) X-100 ™):HA AFLUFDA035 122 455 3.73 AFLUFDA036 139 523 3.76AFLUFDA037 131 475 3.62 AFLUIDA041 148 415 2.80 AFLUIDA042 150 376 2.50AFLUIDA043 156 418 2.68

TABLE 9 Testing of HA content by SRD using NIBSC standards (μg/ml) LotNo 0-day 2 months 4 months 6 months 9 months 12 months AFLUFDA035 122134 128 134 128 126 AFLUFDA036 139 156 135 147 142 153 AFLUFDA037 131138 125 136 135 144

TABLE 10 Testing of HA content by SRD using CBER standards (μg/ml) LotNo 0-day 2 months 4 months 6 months 9 months 12 months AFLUIDA041 152145 163 166 153 162 AFLUIDA042 162 164 170 170 163 161 AFLUIDA043 157166 144 176 168 167

Example 4: Preclinical Studies in Mice

In Vivo Potency Mice Methods

Inhibition of Haemagglutination Assay.

Anti-hemagglutinin antibody titers to the a/California/7/2009-X179Ainfluenza virus strains were determined using the hemagglutinationinhibition test (HI). The principle of the HI test is based on theability of specific anti-Influenza antibodies to inhibithemagglutination of chicken red blood cells (RBC) by influenza virushemagglutinin (HA). Heat inactivated sera were previously treated byKaolin and chicken RBC to remove non-specific inhibitors. Afterpre-treatment, two-fold dilutions of sera were incubated with 4hemagglutination units of each influenza strain. Chicken red blood cellswere then added and the inhibition of agglutination was scored. Thetiters were expressed as the reciprocal of the highest dilution of serumthat completely inhibited hemagglutination. As the first dilution ofsera was 1:20, an undetectable level was scored as a titer equal to 10.

In Vivo Potency Test in Mice Immunized with the Non-AdjuvantedA/California/7/2009-X179A Split Vaccine

Experimental Design and Objective

Treatment/Group (Table 11)

Groups of 10 adult female BALB/c mice were vaccinated intraperitoneallywith two doses of A/California/7/2009-NYMC X-179A split vaccine in atotal volume of 1 ml. Mice were immunized with formulations containing afull Human dose (15 μg HA) or fraction of human dose (3.75, 1.9, 0.46and 0.115 μg of HA) of A/California/7/2009 split vaccine withoutaddition of t-octylphenoxypolyethoxyethanol (TRITON X-100™) in the bulkt-octylphenoxypolyethoxyethanol (TRITON X-100™)/HA ratio of 1) or withaddition of t-octylphenoxypolyethoxyethanol (TRITON X-100™) in the bulkt-octylphenoxypolyethoxyethanol (TRITON X-100™)/HA ratio of 2.83).

Mice were immunized with formulations prepared extemporaneously (TO) orwith similar formulations stored 30 days at 30° C. (acceleratedstability assumed to mimic a long term stability at 4° C.).

A/California/7/09 split vaccine without addition of octoxynol 10: LotAFLSFDA048 at 53 μg HA/ml.

A/California/7/09 split vaccine with addition of octoxynol 10: LotAFLSFDA107 at 180 μg HA/ml.

TABLE 11 A/California/7/2009 (HA/ Dilution Gr Antigen/Formulation dose)step Other treatment 1 A/California without t- 15 Full HD Days 0 and 14octylphenoxypolyethoxyethanol (TRITON X-100 ™) (Lot AFLSFDA048) 2 3.75 ¼Days 0 and 14 3 1.9 ⅛ Days 0 and 14 4 0.46 1/32 Days 0 and 14 5 0.1151/128 Days 0 and 14 6 A/California with t- 15 Full HD Days 0 and 14octylphenoxypolyethoxyethanol (TRITON X-100 ™) (Lot AFLSFDA107) 7 3.75 ¼Days 0 and 14 8 1.9 ⅛ Days 0 and 14 9 0.46 1/32 Days 0 and 14 10 0.1151/128 Days 0 and 14 11 Dilution buffer A

Preparation of the Vaccine Formulations

Polyoxyethylene sorbitan monooleate (TWEEN-80™, or POLYSORBATE 80™),t-octylphenoxypolyethoxyethanol (TRITON X-100™) and Thiomersal are addedto buffer A (130 mM NaCl, 2.68 mM KCl, 0.49 mM MgCl2. 6H2O, 7.26 mMNa2HPO4.12 H2O, 2.74 mM KH2PO4) in order to reach a final concentrationinto the vaccine of 115.4 μg/ml of polyoxyethylene sorbitan monooleate(TWEEN-80™, or POLYSORBATE 80™), 15 μg/ml oft-octylphenoxypolyethoxyethanol (TRITON X-100™) and of 10 μg/ml ofThiomersal. The quantities used are calculated taking into account thequantities already present in the strains. The premix are mixed 15 to 45minutes on an orbital shaking table at room temperature before theaddition of 15 μg of A/California split vaccines. After the addition ofthe split vaccines, the formulations are mixed 15 to 45 min on anorbital shaking table at room temperature.

Part of the full dose formulations are extemporaneously diluted 1/4,1/8, 1/32 and 1/128-fold in the same buffer A (see above) by serialdilutions. The full dose and the dilutions are injected within the hourfollowing the end of their preparation.

Another part of the full dose formulation is incubated 30 days at 30° C.and extemporaneously diluted and injected.

Read-Outs

The humoral immune response to vaccination was measured 14 days afterthe second immunization (Day 28), on 10 mice/group. Serum samples weretested by the hemagglutination inhibition assay (HI).

Results

Humoral Immune Responses

The results are shown in FIGS. 4 and 5.

Two immunizations are required in mice immunized with the non-adjuvantedvaccine in order to induce HI titers >40.

With the process without addition of t-octylphenoxypolyethoxyethanol(TRITON X-100™) (FIG. 4), only the mice immunised with the full humandose (15 μg HA) of the non-adjuvanted vaccine stored 30 days at 30° C.showed similar immune response compared to the mice immunised with thenon-adjuvanted vaccine prepared extemporaneously.

With the addition of t-octylphenoxypolyethoxyethanol (TRITON X-100™) inthe process (FIG. 5), similar immune responses were observed betweenmice immunized with the full human dose (15 μg HA) and 1/4 human dose ofthe non-adjuvanted vaccine stored 30 days at 30° C. and mice immunizedwith the non-adjuvanted vaccine prepared extemporaneously.

Conclusion

Compared to the immune response induced in mice immunised with thenon-adjuvanted vaccine prepared extemporaneously, the non-adjuvantedvaccine stored 30 days at 30° C. showed higher stability with theaddition of t-octylphenoxypolyethoxyethanol (TRITON X-100™) compared tothe non-adjuvanted vaccine formulated without addition oft-octylphenoxypolyethoxyethanol (TRITON X-100™).

In Vivo Potency Test in Mice Immunized with AS03A-AdjuvantedA/California/7/2009-X179A Split Vaccine

Experimental Design and Objective

Treatment/Group (Table 12)

Groups of 10 adult female BALB/c mice were immunised intraperitoneallywith two doses of A/California/7/2009-NYMC X-179A split vaccineadjuvanted with AS03A in a total volume of 1 ml. A Human dose (HD) wasconsidered at 15 μg HA and containing 11.8 mg alpha-tocopherol (VitaminE).

Mice were immunised with a half Human dose of adjuvanted vaccine and2-fold or 4-fold step dilution of vaccine (7.5, 3.75, 1.9, 0.46 and0.115 μg HA) without the addition of t-octylphenoxypolyethoxyethanol(TRITON X-100™) in the bulk (t-octylphenoxypolyethoxyethanol (TRITONX-100™)/HA ratio of 1) or with the addition oft-octylphenoxypolyethoxyethanol (TRITON X-100™) in the bulk(t-octylphenoxypolyethoxyethanol (TRITON X-100™)/HA ratio of 2.83).

Mice were immunized with formulations prepared extemporaneously (TO) orwith similar formulations stored 30 days at 30° C.

Split A/California virus antigen without additional octoxynol: LotAFLSFDA048-53 μgHA/ml

Split A/California virus antigen containing additional octoxynol 10: LotAFLSFDA107-180 μgHA/ml

TABLE 12 A/California/ 7/2009 (Conc Gr Antigen/Formulation HA/dose)Dilution step Other treatment A/California without t-   15 μg Full HDoctylphenoxy- polyethoxyethanol (TRITON X- 100 ™)-AS03A (Lot AFLSFDA048)1  7.5 μg ½ Days 0 and 14 2 3.75 μg ¼ Days 0 and 14 3  1.9 μg ⅛ Days 0and 14 4 0.46 μg 1/32 Days 0 and 14 5 0.115 μg  1/128 Days 0 and 14A/California with   15 μg Full HD t-octylphenoxy- polyethoxyethanol(TRITON X- 100 ™)-AS03A (Lot AFLSFDA107) 6  7.5 μg ½ Days 0 and 14 73.75 μg ¼ Days 0 and 14 8  1.9 μg ⅛ Days 0 and 14 9 0.46 μg 1/32 Days 0and 14 10 0.115 μg  1/128 11 Dilution buffer A

Data obtained with full human dose and 1/2 human dose ofAS03A-adjuvanted A/California/7/09 vaccine were not included due to highlevel of mortality (>50%) in mice immunised by intraperitoneal route.

Preparation of the Vaccine Formulations.

Polyoxyethylene sorbitan monooleate (TWEEN-80™ or POLYSORBATE 80™),t-octylphenoxypolyethoxyethanol (TRITON X-100™) and Thiomersal are addedto buffer A (130 mM NaCl, 2.68 mM KCl, 0.49 mM MgCl2. 6H2O, 7.26 mMNa2HPO4.12 H2O, 2.74 mM KH2PO4) in order to reach a final concentrationinto the vaccine of 115.4 μg/ml of polyoxyethylene sorbitan monooleate(TWEEN-80™ or POLYSORBATE 80™), 15 μg/ml oft-octylphenoxypolyethoxyethanol (TRITON X-100™) and of 10 μg/ml ofThiomersal. The quantities used are calculated to take into account thequantities already present in the strains. The premix are mixed 15 to 45minutes on an orbital shaking table at room temperature before theaddition of 15 μg of A/California split vaccines. After the addition ofthe split vaccines, the formulations are mixed for 15 to 45 min on anorbital shaking table at room temperature. The oil-in-water emulsion isthen added in order to reach a final concentration of 10.69 mg squalene,11.86 mg alpha-tocopherol and 4.86 mg polyoxyethylene sorbitanmonooleate (TWEEN-80™ or POLYSORBATE 80™) per ml. The vaccine is mixed15 to 45 min on an orbital shaking table at room temperature.

Part of the full dose formulations are extemporaneously diluted 1/2,1/4, 1/8, 1/32 and 1/128-fold in buffer A by serial dilutions. The fulldose and the dilutions are injected within the hour following the end oftheir preparation.

Another part of the full dose formulations is incubated 30 days at 30°C. and extemporaneaously diluted and injected.

Read-Outs

The humoral immune response to vaccination was measured 14 days afterthe first (Day 14) and the second immunization (Day 28) on 10mice/group. Serum samples were tested by the inhibition hemagglutinationassay (HI).

Results

Humoral Immune Responses

The results are shown in FIGS. 6 and 7, 14 days after the first dose,and in FIGS. 8 and 9, 14 days after the second dose.

HI titers above 40 were observed after one dose of AS03A-adjuvantedvaccine prepared extemporaneously. After one dose of AS03A-adjuvantedvaccine, and whether t-octylphenoxypolyethoxyethanol (TRITON X-100™) waspresent (FIG. 7) or not (FIG. 6), significant lower HI titers wereobserved after storage 30 days at 30° C. compared to the immune responseinduced in mice immunized with the AS03A-adjuvanted vaccine preparedextemporaneously.

Higher HI titres were observed in mice immunized with two doses ofAS03A-adjuvanted vaccines compared to the immune response induced inmice immunized with a single dose of AS03A-adjuvanted vaccine (FIGS. 8and 9).

After administration of two doses of AS03A-adjuvanted vaccines preparedwithout the addition of t-ocylphenoxypolyethoxyethanol (TRITON X-100™)(FIG. 8), lower HI titers were observed in mice immunized with thevaccine stored 30 days at 30° C. compared to mice immunized with thevaccine prepared extemporaneously.

With the addition of t-octylphenoxypolyethoxyethanol (TRITON X-100™) inthe process (FIG. 9), similar immune responses were observed in miceimmunized with the adjuvanted vaccine stored 30 days at 30° C. and theadjuvanted vaccine prepared extemporaneously.

Conclusion

Compared to the immune response induced in mice immunized with theadjuvanted vaccine prepared extemporaneously, the adjuvanted vaccinestored 30 days at 30° C. showed higher stability with the addition oft-octylphenoxypolyethoxyethanol (TRITON X-100™) compared to theadjuvanted vaccine prepared without the addition oft-octylphenoxypolyethoxyethanol (TRITON X-100™).

General Conclusion

Vaccination of mice with non-adjuvanted or AS03A-adjuvantedA/California/7/09 split vaccines produced with a process containinghigher concentration of t-octylphenoxypolyethoxyethanol (TRITON X-100™)resulted in higher humoral immune responses (HI titers) compared tovaccines prepared without the addition oft-octylphenoxypolyethoxyethanol (TRITON X-100™). These data demonstratedan improved stability of the split vaccine, adjuvanted or not withAS03A, when this vaccine is prepared with an addition oft-octylphenoxypolyethoxyethanol (TRITON X-100™) during the process.

Example 5: A Phase III, Double-Blind, Randomized, Study in Adults AgedBetween 18 and 60 Years

Introduction and Study Design

A phase III, double-blind, randomized, study was conducted in adultsaged between 18 and 60 years to assess the immunological non-inferiorityof two manufacturing processes of the A/California/7/2009 (H1N1)v-likeantigen adjuvanted with AS03A.

Four parallel groups (1:1:1:1) with approximately 300 subjects in totalreceiving the following vaccines IM:

-   -   D-INI-1D group: 75 subjects receiving one dose of initial        process-manufactured Flu D-PAN H1N1 candidate vaccine adjuvanted        with AS03A    -   D-INI-2D group: 75 subjects receiving two doses of initial        process-manufactured Flu D-PAN H1N1 candidate vaccine adjuvanted        with AS03A    -   D-NEW-1D: 75 subjects receiving one dose of new        process-manufactured Flu D-PAN H1N1 candidate vaccine adjuvanted        with AS03A    -   D-NEW-2D: 75 subjects receiving two doses of new        process-manufactured Flu D-PAN H1N1 candidate vaccine adjuvanted        with AS03A

Schedule: One or two intramuscular (IM) injections, only one for the twogroups 1 (Day 0) or two for the two groups 2 (Day 0 and Day 21), bloodsample collection at day 0, day 21, day 42, day 182 and day 364postvaccination.

Study Objectives

Immunological non-inferiority (in terms of vaccine-homologous virus H1N1HI antibody GMTs—Haemagglutination Inhibition (HI) antibodies) of thenew process-manufactured A/California/7/2009(H1N1)v-like antigenadjuvanted with AS03A was compared to the initial process-manufacturedA/California/7/2009 (H1N1)v-like antigen adjuvanted with AS03A, 21 daysafter first vaccination in healthy subjects aged 18 to 60 years.Non-inferiority is considered to be reached if the upper limit of thetwo-sided 95% CI for the ratio of GMT between the initialprocess-manufactured vaccine and (over) new process-manufactured vaccineis less than or equal to 2 in terms of HI antibody titre againstA/California/7/2009 (H1N1)v-like. Geometric mean titres (GMTs) of HIantibody titres 21 days after first dose of vaccine (Day 21) and 21 daysafter second vaccination have also been measured. Additional parameterssuch as seroconversion rates (SCR—defined as the percentage of vaccineeswho have either a prevaccination titre <1:10 and a post-vaccinationtitre ≥1:40 or a pre-vaccination titre ≥1:10 and at least a four-foldincrease in post-vaccination titre), Seroconversion factors (SCF—definedas the fold increase in serum HI GMTs post-vaccination compared topre-vaccination), seroprotection rates (SPR—defined as the percentage ofvaccinees with a serum HI titre ≥1:40 that usually is accepted asindicating protection), and neutralizing antibodies are also reportedbelow.

Antigen Preparation

The only difference between the two processes D-INI and D-NEW is theaddition of t-octylphenoxypolyethoxyethanol (TRITON X-100™), immediatelyafter splitting of the virus. This helps preventing aggregation of theantigen and loss of antigen during the final sterile filtration step.FIG. 1 shows an overview of the method of preparation of the split,inactivated H1N1v bulk preparation for D-INI (left column) and D-NEW(middle column).

Basically, the manufacturing process of the bulks can be divided in fourmain parts and is largely based on the process described in Example 5aof WO 02/097072 (U.S. Pat. No. 7,316,813B2) with the omission of theaddition of alpha-tocopherol succinate stabiliser:

1. Propagation of the working seed in fertilized hen's eggs, harvestingand pooling of infected allantoic fluids so as to obtain the “crudewhole virus bulk” (step 1).

2. Purification of each virus strain leading to the “purified wholevirus bulk” (steps 2-6).

3. Splitting of the purified monovalent whole virus bulk with sodiumdeoxycholate resulting in the “purified split virus bulk” (steps 7-8/1).

4. Inactivation of the purified monovalent split virus bulk in two stepsby incubation with sodium deoxycholate and with formaldehyde, followedby ultrafiltration and sterile filtration, in order to obtain the“purified inactivated split virus bulk”, or “Bulk” (steps 8/2-9).

The purified split virus bulk (fraction 7/2 in FIG. 1) of the D-NEWprocess is then subjected to the following treatment. Prior tofiltration, t-octylphenoxypolyethoxyethanol (TRITON X-100™) is added (soas to reach 0.25% final concentration in the filtrate). Fraction 7/2 isgradually filtered down to a 0.45 μm or higher filter membrane, followedby a second membrane filtration step with a pore size of 0.45 μm orlower, with the aim of removing bioburden. The filtrate is then brieflysonicated and filtered through a membrane of pore size 0.45 μm or below.At the end of the filtration, the filters are rinsed with phosphatebuffer containing 0.025% Tween-80. As a result of the filtration andrinsing, the final volume of the filtrate is 5-fold the originalfraction 7/2 volume.

Thiomersal is added to the formulation (in both processes), present in10-doses presentation, at a concentration of 5 μg per dose.

Vaccine Composition

The HA content per dose of vaccine has been fixed at 3.75 μg per 0.5 mldose and the vaccine after reconstitution contains some excipients suchas polyoxyethylene sorbitan monooleate (TWEEN-80™, or POLYSORBATE 80™)and t-octylphenoxypolyethoxyethanol (TRITON X-100™) to an amount of≥28.75 μg and 22.5 μg per 0.5 ml dose respectively. AS03A adjuvant is anoil-in-water emulsion adjuvant system containing 11.86 mg tocopherol,10.69 mg squalene and 4.86 mg polyoxyethylene sorbitan monooleate(TWEEN-80™, or POLYSORBATE 80™) per 0.5 ml vaccine dose (WO2006/100109).The reconstituted vaccine is made by mixing the adjuvant O/W emulsionwith the antigen suspension. The composition of the reconstitutedvaccine also contains 5 μg thiomersal per 0.5 ml dose.

The antigen lots used in the study were:

-   -   Lot no for H1N1 (new process D-NEW)=DFLSA014A; lot no for the        adjuvant AA03A209C; the t-octylphenoxypolyethoxyethanol (TRITON        X-100™) content is 22.5 μg/0.25 mL of antigen suspension    -   Lot no for H1N1 (initial process D-INI)=DFLSA013A; lot no for        the adjuvant AA03A209C; the t-octylphenoxypolyethoxyethanol        (TRITON X-100™) content is 3.75 μg/0.25 mL (ratio of 1:1 to the        HA content) of antigen suspension

The content in Tween-80 for the H1N1 antigen suspension is >=28.75μg/0.25 mL of antigen suspension (target formulation of 115 μg/ml) forboth antigen suspensions but does not exceed 55 μg/0.25 mL (220 μg/mL).The composition of the antigen suspension is in Table 13.

TABLE 13 Composition of inactivated split virion H1N1 antigen componentQuantity per Quantity per 0.25 ml - 0.25 ml - DFLSA013A (initialDFLS014A Ingredients process) (adapted process) Unit Purified antigenfractions of inactivated split 3.75 3.75 μg HA virionA/California/7/2009 (H1N1)v NYMC X-179A Polyoxyethylene sorbitanmonooleate ≥28.75 ≥28.75 μg (TWEEN-80 ™, or POLYSORBATE 80 ™)t-octylphenoxypolyethoxyethanol 3.75 22.5 μg (TRITON X-100 ™) Sodiumchloride 1.92 1.92 mg Disodium phosphate 0.26 0.26 mg Potassiumdihydrogen phosphate 0.094 0.094 mg Potassium chloride 0.050 0.050 mgMagnesium chloride 0.012 0.012 mg Thiomersal 5 5 μg Water for injectionsq.s. ad. 0.25 0.25 ml

Immunogenicity Results—Humoral Immune Response

Non-Inferiority of the New Process-Manufactured A/California/7/2009(H1N1)v-Like Antigen Adjuvanted with AS03A Vaccine Compared to theInitial Process-Manufactured A/California/7/2009 (H1N1)v-Like AntigenAdjuvanted with AS03A Vaccine (GMT).

The new process-manufactured A/California/7/2009 (H1N1)v-like antigenadjuvanted with AS03A vaccine was shown to be non-inferior to theinitial process-manufactured A/California/7/2009 (H1N1)v-like antigenadjuvanted with AS03A vaccine, both in terms of GMT ratios at Day 21(Table 14) and in terms of seroconversion rate difference at Day 21(Table 15).

TABLE 14 Adjusted GMT ratios between D-INI and D-NEW for HI antibodiesat Day 21 (ATP cohort for immunogenicity) Adjusted GMT ratio GroupAdjusted Group Adjusted 95% CI description N GMT description N GMT Ratioorder Value LL UL D-INI 144 437.9 D-NEW 146 392.2 D-INI/D-NEW 1.12 0.891.40 1. D-NEW = Subjects receiving the new process-manufactured FluD-PAN H1N1 vaccine with adjuvant 2. D-INI = Subjects receiving theinitial process-manufactured Flu D-PAN H1N1 vaccine with adjuvant 3.Adjusted GMT = geometric mean antibody titer adjusted for baseline titer4. N = Number of subjects with both pre- and post-vaccination resultsavailable 5. 95% CI = 95% confidence interval for the adjusted GMT ratio(Ancova model: adjustment for baseline titer-pooled variance); LL =lower limit, UL = upper limit

TABLE 15 The difference between D-INI and D-NEW seroconversion rate atDay 21 (ATP cohort for immunogenicity) Difference in vaccine responserate Pre- (D-INI minus D-NEW) vaccination D-INI D-NEW 95% CI Antibodystatus N n % N n % % LL UL Flu Total 144 138 95.8 146 141 96.6 −0.74−5.82 4.16 A/CAL/7/09.HA1 Ab (1/DIL) 1. D-NEW = Subjects receiving thenew process-manufactured Flu D-PAN H1N1 vaccine with adjuvant 2. D-INI =Subjects receiving the initial process-manufactured Flu D-PAN H1N1vaccine with adjuvant 3. N = number of subjects with pre- andpost-vaccination results available 4. n/% = number/percentage ofsubjects with a vaccine response 5. 95% CI = Standardized asymptotic 95%confidence interval; LL = lower limit, UL = upper limit

HI Geometric Mean Titres (GMT)

The GMT's for HI antibodies with 95% CI are shown in Table 16. At Day 0,seropositivity rates were 43.2% to 47.2%. Before vaccination, GMT valueswere low and ranged between 9.6 and 10.2. At Day 21, seropositivityrates increased to 100% in both groups (D-INI and D-NEW). The amplitudeof the observed HI response was similar in both groups (GMTs of 388.8for the D-NEW group versus 441.8 for the D-INI group).

TABLE 16 Seropositivity rates and GMTs for HI antibodies at Day 0 andDay 21 (ATP cohort for immunogenicity). >=10 1/DIL GMT 95% CI 95% CIAntibody Group Timing N n % LL UL value LL UL Min Max Flu D- PRE 146 6343.2 35.0 51.6 9.6 8.3 11.2 <10.0 640.0 A/CAL/7/09.HA1 NEW PI(D21) 146146 100 97.5 100 388.8 331.3 456.3 40.0 2560.0 Ab D-INI PRE 144 68 47.238.9 55.7 10.2 8.7 12.0 <10.0 640.0 PI(D21) 144 144 100 97.5 100 441.8372.4 524.0 20.0 5120.0 1. D-NEW = Subjects receiving the newprocess-manufactured Flu D-PAN H1N1 vaccine with adjuvant 2. D-INI =Subjects receiving the initial process-manufactured Flu D-PAN H1N1vaccine with adjuvant 3. GMT = geometric mean antibody titer calculatedon all subjects 4. N = number of subjects with available results 5. n/%= number/percentage of subjects with titer within the specified range 6.95% CI = 95% confidence interval; LL = Lower Limit, UL = Upper Limit 7.MIN/MAX = Minimum/Maximum 8. PRE = visit 1 Day 0 9. PI(D21) = visit 2Day 21

Seroprotection Rates, Seroconversion Rates and Seroconversion Factors ofAnti-HI Ab Titres

Results are presented in Table 17 for seroprotection rates (SPR), Table18 for seroconversion rates (SCR) and Table 19 for seroconversionfactors (SCF).

SPR were shown to be within the same range and met CHMP (mean >70) inboth groups. SCR induced by the new process-manufactured Flu D-PAN H1N1vaccine and the initial process-manufactured Flu D-PAN H1N1 vaccine werewithin the same range and both met CHMP criteria (mean >40). For bothgroups SCF were within the same range and were above the CHMP criteria(>2.5).

TABLE 17 Seroprotection rates (SPR) for HI antibodies at Day 0 and Day21 (ATP cohort for immunogenicity) SPR 95% CI Strain Group Timing N n %LL UL Flu A/CAL/7/ D-NEW PRE 146 17 11.6 6.9 18.0 09.HA1 Ab PI(D21) 146146 100 97.5 100 D-INI PRE 144 22 15.3 9.8 22.2 PI(D21) 144 142 98.695.1 99.8 1. D-NEW = Subjects receiving the new process-manufactured FluD-PAN H1N1 vaccine with adjuvant 2. D-INI = Subjects receiving theinitial process-manufactured Flu D-PAN H1N1 vaccine with adjuvant 3. N =Number of subjects with available results 4. n/% = Number/percentage ofseroprotected subjects (HI titer >=40 1/DIL) 5. 95% CI = 95% confidenceinterval, LL = Lower Limit, UL = Upper Limit 6. PRE = visit 1 Day 0 7.PI(D21) = visit 2 Day 21

TABLE 18 Seroconversion rate (SCR) for HI antibodies at Day 21 (ATPcohort for immunogenicity) SCR 95% CI Strain Group Timing N n % LL ULFlu A/CAL/7/ D-NEW PI(D21) 146 141 96.6 92.2 98.9 09.HA1 Ab D-INIPI(D21) 144 138 95.8 91.2 98.5 1. D-NEW = Subjects receiving the newprocess-manufactured Flu D-PAN H1N1 vaccine with adjuvant 2. D-INI =Subjects receiving the initial process-manufactured Flu D-PAN H1N1vaccine with adjuvant 3. N = Number of subjects with pre- andpost-vaccination results available 4. n/% = Number/percentage ofseroconverted subjects 5. 95% CI = 95% confidence interval, LL = LowerLimit, UL = Upper Limit 6. PI(D21) = visit 2 Day 21

TABLE 19 Seroconversion factor (SCF) for HI antibody titer at Day 21(ATP cohort for immunogenicity) SCF 95% CI Vaccine strain Group Timing NValue LL UL Flu A/CAL/7/09.HA1 Ab D-NEW PI(D21) 146 40.4 33.3 49.1(1/DIL) D-INI PI(D21) 144 43.4 35.9 52.4 1. D-NEW = Subjects receivingthe new process-manufactured Flu D-PAN H1N1 vaccine with adjuvant 2.D-INI = Subjects receiving the initial process-manufactured Flu D-PANH1N1 vaccine with adjuvant 3. N = Number of subjects with pre- andpost-vaccination results available 4. SCF = Seroconversion Factor orgeometric mean ratio (mean[log10(POST/PRE)]) 5. 95% CI = 95% confidenceinterval, LL = Lower Limit, UL = Upper Limit 6. PI(D21) = visit 2 Day 21

Immunological Response Per Age Strata

Results are presented in Table 20 for seropositivity, GMT,seroprotection rates, seroconversion rates and for seroconversionfactors, overall and by age stratum (18-40 years and 41-60 years).

Overall as per age strata, at Day 21, immune responses in subjects fromD-INI and D-NEW groups both exceeded all CHIMP regulatory acceptancecriteria for influenza vaccines in adults. All tested parameters can beconsidered as similar between both groups.

TABLE 20 H1N1 HI antibodies against A/California/7/2009 (H1N1)v-likestrain by age strata and overall. ≥10 1/DIL GMT SPR SCR^(#) SCF 95% CI95% CI 95% CI 95% CI 95% CI Timing N % LL UL value LL UL % LL UL % LL ULvalue LL UL Overall D-NEW group PRE 146 43.2 35.0 51.6 9.6 8.3 11.2 11.66.9 18.0 — — — — — — PI(D21) 146 100 97.5 100 388.8 331.3 456.3 100 97.5100 96.6 92.2 98.9 40.4 33.3 49.1 18-40 years stratum D-NEW group PRE 7445.9 34.3 57.9 10.3 8.2 12.9 12.2 5.7 21.8 — — — — — — PI(D21) 74 10095.1 100 456.8 373.4 558.8 100 95.1 100 97.3 90.6 99.7 44.4 34.6 57.141-60 years stratum D-NEW group PRE 72 40.3 28.9 52.5 9.0 7.4 11.0 11.14.9 20.7 — — — — — — PI(D21) 72 100 95.0 100 329.5 256.9 422.5 100 95.0100 95.8 88.3 99.1 36.6 27.0 49.7 Overall D-INI group PRE 144 47.2 38.955.7 10.2 8.7 12.0 15.3 9.8 22.2 — — — — — — PI(D21) 144 100 97.5 100441.8 372.4 524.0 98.6 95.1 99.8 95.8 91.2 98.5 43.4 35.9 52.4 18-40years stratum D-INI group PRE 74 45.9 34.3 57.9 10.1 8.0 12.9 16.2 8.726.6 — — — — — — PI(D21) 74 100 95.1 100 564.0 446.9 711.8 100 95.1 10097.3 90.6 99.7 55.6 42.9 72.1 41-60 years stratum D-INI group PRE 7048.6 36.4 60.8 10.2 8.2 12.8 14.3 7.1 24.7 — — — — — — PI(D21) 70 10094.9 100 341.2 267.9 434.6 97.1 90.1 99.7 94.3 86.0 98.4 33.3 25.4 43.6GMT = Geometric Mean Titer; SPR = Seroprotection rate: percentage ofsubjects with antibody titer ≥1:40; SCR = Seroconversion rate:percentage of subjects with antibody titer ≥40 1/DIL after vaccinationfor initially seronegative subjects, or ≥4 fold the pre-vaccinationantibody titer for initially seropositive subjects; SCF = Seroconversionfactor: fold increase in GMTs post-vaccination compared withpre-vaccination; PRE = pre-vaccination; PI(D21) = post-vaccination I atDay 21; LL = Lower Limit; UL = Upper Limit; N = number of subjects withavailable results.

Reactogenicity Results

Solicited local symptoms were reported at similar frequencies in theD-NEW and D-INI groups. Pain at injection site was the most frequentlyreported symptom, while swelling and redness were observed at lowerfrequencies in both groups. Grade 3 solicited local symptoms wereinfrequent and similar in both groups.

With the exception of shivering which was reported with a higherfrequency in the D-INI (27.5%) compared to the D-NEW (18.8%) group,solicited general symptoms were reported at equivalent frequencies inboth groups. Grade 3 related solicited general symptoms were infrequentand similar in both groups (between 0% and 2.0%).

Conclusions

Immunogenicity Conclusions

The immunological non-inferiority objective (in terms of GMTs and SCRs)of the new process-manufactured A/California/7/2009 (H1N1)v-like antigenadjuvanted with AS03A compared to the initial process-manufacturedA/California/7/2009 (H1N1)v-like antigen adjuvanted with AS03A, 21 daysafter first vaccination in healthy subjects aged 18 to 60 years wereboth reached.

The HI humoral immune response observed 21 days following the firstadministration of the influenza pandemic vaccine at 3.75 μg HA antigenmet and exceeded all EMEA/CHMP regulatory acceptance criteria in termsof SCR, SPR and SCF in all age strata and in both groups, havingreceived one dose of either the initial or the new process-manufacturedvaccines.

Furthermore the EMEA/CHMP acceptance criteria in terms of SCR, SPR andSCF are also met and exceeded 21 days after the second vaccination forboth vaccines.

Safety Conclusions

The reactogenicity profiles were comparable in both groups. The mostfrequent solicited AEs were pain at injection site, fatigue, musclesaches and headaches. The individual values of all reactogenicityparameters are similar for D-New and D-Ini vaccines.

Overall Conclusion

The H1N1 candidate vaccine manufactured with the new process andadjuvanted with AS03A elicits an immune response against theA/California/7/2009 (H1N1)v-like strain in adults that is non inferiorto the immune response induced by the vaccine antigen manufacturedaccording to the initial process. Both vaccines demonstrated anacceptable reactogenicity profile, no safety concerns were raised.

We claim:
 1. A split influenza virus preparation or subunit influenzavirus preparation obtained or obtainable by a process comprising thesteps of: (i) providing a whole virus preparation; (ii) splitting thewhole virus preparation in the presence of a first detergent, whereinthe first detergent is not t-octylphenoxypolyethoxyethanol, therebyproducing a split virus preparation; (iii) addingt-octylphenoxypolyethoxyethanol to the split virus preparation, whereint-octylphenoxypolyethoxyethanol is present in an amount of 0.1-0.4%(w/v); and (iv) filtering the split virus preparation in the presence oft-octylphenoxypolyethoxyethanol.
 2. The split influenza viruspreparation or subunit influenza virus preparation of claim 1, whereint-octylphenoxypolyethoxyethanol is present in an amount of 0.25% (w/v).3. The split influenza virus preparation or subunit influenza viruspreparation of claim 1, wherein the first detergent is selected from thegroup consisting of: cetyl trimethyl ammonium bromide (CTAB),laurylsulfate, taurodeoxycholate, polyoxyethylene sorbitan monooleateand sodium deoxycholate.
 4. The split influenza virus preparation orsubunit influenza virus preparation of claim 1, wherein the firstdetergent is sodium deoxycholate.
 5. A process for preparing apharmaceutical or immunogenic composition, comprising the steps of: (a)providing a split influenza virus preparation or subunit influenza viruspreparation by a process comprising the steps of: (i) providing a wholevirus preparation; (ii) splitting the whole virus preparation in thepresence of a first detergent, wherein the first detergent is nott-octylphenoxypolyethoxyethanol, thereby producing a split viruspreparation; (iii) adding t-octylphenoxypolyethoxyethanol to the splitvirus preparation, wherein t-octylphenoxypolyethoxyethanol is present inan amount of 0.1-0.4% (w/v); and (iv) filtering the split viruspreparation in the presence of t-octylphenoxypolyethoxyethanol; and (b)admixing said split influenza virus preparation or subunit influenzavirus preparation with a pharmaceutically acceptable carrier to preparethe pharmaceutical or immunogenic composition.
 6. The process of claim5, wherein t-octylphenoxypolyethoxyethanol is present in an amount of0.25% (w/v).
 7. The process of claim 5, wherein the first detergent isselected from the group consisting of: cetyl trimethyl ammonium bromide(CTAB), laurylsulfate, taurodeoxycholate, polyoxyethylene sorbitanmonooleate and sodium deoxycholate.
 8. The process of claim 5, whereinthe first detergent is sodium deoxycholate.
 9. The process of claim 5further comprising a step wherein the split influenza virus preparationor subunit influenza preparation of step (a) is diluted to reach thedesired HA concentration before step (b).
 10. A pharmaceutical orimmunogenic composition obtained or obtainable by the process of claim5.
 11. The pharmaceutical or immunogenic composition of claim 10additionally comprising an adjuvant.
 12. The pharmaceutical orimmunogenic composition of claim 11 wherein the adjuvant is an oil inwater emulsion.
 13. A method of treating or preventing a disease causedby influenza, the method comprising administering the pharmaceutical orimmunogenic composition of claim 10 to a human subject in need thereof.14. A vaccine comprising one or more split influenza virus preparationsor subunit influenza virus preparations prepared according to a processcomprising the steps of: (i) providing a whole virus preparation; (ii)splitting the whole virus preparation in the presence of a firstdetergent, wherein the first detergent is nott-octylphenoxypolyethoxyethanol, thereby producing a split viruspreparation; (iii) adding t-octylphenoxypolyethoxyethanol to the splitvirus preparation, wherein t-octylphenoxypolyethoxyethanol is present inan amount of 0.1-0.4% (w/v); and (iv) filtering the split viruspreparation in the presence of t-octylphenoxypolyethoxyethanol.
 15. Thevaccine of claim 14, wherein t-octylphenoxypolyethoxyethanol is presentin an amount of 0.25% (w/v).
 16. The vaccine of claim 14, wherein thefirst detergent is selected from the group consisting of: cetyltrimethyl ammonium bromide (CTAB), laurylsulfate, taurodeoxycholate,polyoxyethylene sorbitan monooleate and sodium deoxycholate.
 17. Thevaccine of claim 14, wherein the first detergent is sodium deoxycholate.